An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms
- Baylor College of Medicine, United States
- Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, United States
- Howard Hughes Medical Institute, Harvard Medical School, United States
- Harvard Medical School, United States
- Howard Hughes Medical Institute, United States
- Howard Hughes Medical Institute, Baylor College of Medicine, United States
- Howard Hughes Medical Institute, Carnegie Institution for Science, United States
Figures
Figure 1
ssDNA pipeline is faster than cloning CRIMIC constructs.
(A) Schematic of PCR-based generation of drop-in ssDNA constructs. Gray boxes, UTRs; orange boxes, coding exons; yellow line, coding introns; black line, outside coding introns and exons. (B) …
Figure 2 with 3 supplements
ssDNA homology donors are effective in S2 cells to tag organelles.
(A) Schematic of drop-in cassette encoding for sfGFP artificial exon. Size of the construct including the homology arms is indicated on the right. sfGFP: superfolderGFP; SA: Splice Acceptor of mhc; …
Figure 2—figure supplement 1
FACS data of control cells (left) and ssDNA knock-in cells (right).
All cell lines express mCherry::Clic, which is present in the parental cell line and thus in these derivatives. Single cell clones with GFP expression levels greater than 2 × 102 were retained.
Figure 2—figure supplement 2
Detection of subcellular localization of GFP tagged proteins in S2 cells.
Left panels, confocal fluorescence detection of GFP fusion proteins in single-cell isolated clones. The specific proteins tagged by GFP knock-in are indicated. Center panels, confocal fluorescence …
Figure 2—figure supplement 3
Western blot analysis of tagged proteins observed in S2R+ cells.
Western blot analysis of selected clones using anti-GFP antibody shows expected protein sizes.
Figure 3
ssDNA constructs are not as efficient as double stranded CRIMIC constructs for fly transformation.
(A) Schematic of drop-in cassette used for fly transformation. Size of the construct including homology arms is indicated on the right. (B) Injection results for the 10 genes selected for comparison …
Figure 4 with 1 supplement
Double stranded DNA synthetic constructs are efficient for fly transformation.
(A) Schematic of synthesized plasmid drop-in donors. mPA indicates minipolyA. (B) Schematic of the donor plasmid followed by linearization by Cas9 in germ cells and integration of donors in vivo. …
Figure 4—figure supplement 1
Schematic and crossing scheme for CRISPR gRNA-based dominant marker strategy.
(A) Schematics of the action of gRNA1-based detection of dominant marker. (B) Crossing scheme for screening the transgenics using U6gRNA1 as dominant marker. The crosses for a second chromosome …
Figure 5
T2A-miniGAL4 gene trap cassette is mutagenic and expresses an active GAL4.
(A) Western blot and quantification of the level of ACOX1 protein in flies homozygous for SA-T2A-miniGAL4-minipolyA, SA-3XSTOP-minipolyA or CRIMIC construct for CG5009. ****p<0.0001, *p<0.01. mPA1 …
Figure 6 with 1 supplement
Single step cloning method allows efficient insertion of the CRIMIC cassette in coding introns.
(A) Schematic of a single step cloning vector pUC57. LHA Left Homology Arm, RE1 Restriction Enzyme 1, RE2 restriction Enzyme 2, RHA Right Homology Arm. (B) Injection results for the seven genes …
Figure 6—figure supplement 1
Comparison of expression domain obtained by T2A-miniGAL4 and drop-in int100-CRIMIC.
Third instar larval brain expression domain of Khc as determined by crossing conventional T2A-miniGAL4 or drop-in int100-CRIMIC flies to UAS-NLS-mCherry reporter lines. Scale bars are 100 µm.
Videos
Video 1
Polo-sfGFP knock-in in S2R+ cells through ssDNA drop-in faithfully reports dynamic localization of Polo throughout the cell cycle.
https://doi.org/10.7554/eLife.51539.008Tables
Table 1
Summary of ssDNA drop-in mediated homologous recombination in S2R+ cells
https://doi.org/10.7554/eLife.51539.007| Organelle | Fly protein | clones obtained | population imaged | clones imaged | insertion sequence verified | Immunostained | Correct GFP localization | DGRC stock# | S2R+ expression (modENCODE RPKM) |
|---|---|---|---|---|---|---|---|---|---|
| Autophagosomes | Atg8a | 14 | ✔ | ✔ | N | N | N | N | 153 |
| Autophagosomes/aggregates | Ref(2)P | 9 | ✔ | ✔ | Y | a-Ref2P, a-FK2 | N | N | 138 |
| Endoplasmic reticulum (ER) | Calnexin99A | 16 | ✔ | ✔ | Y | a-Cnx99a | Y | 273 | 235 |
| Endoplasmic reticulum (ER), transitional | Sec23 | 30 | ✔ | ✔ | Y | N | * | 294 | 101 |
| Endosomes, early | Rab5 | 9 | ✔ | ✔ | N | N | N | N | 98 |
| Endosomes, recycling | Rab11 | 23 | ✔ | ✔ | Y | N | * | 274 | 302 |
| G-Bodies (cytoplasmic puncta) | Pfk | 14 | ✔ | ✔ | N | N | N | N | 23 |
| Golgi (cis-Golgi) | Gmap | 10 | ✔ | ✔ | Y | a-GMAP | Y | 276, 277 | 15 |
| Golgi (trans-Golgi) | Sec71 | 10 | ✔ | ✔ | N | N | N | N | 8 |
| Golgi (trans-Golgi) | Golgin245 | 1 | ✔ | ✔ | Y | a-Golgin245 | Y | 280 | 27 |
| Kinetochore | Polo | 2 | ✔ | ✔ | Y | N | Y | 275 | 50 |
| Lipid droplets | Seipin | 12 | ✔ | ✔ | N | N | N | N | 9 |
| Lysosomes | spin | 2 | ✔ | ✔ | Y | a-Arl8 | Y | 293 | 112 |
| Lysosomes | Arl8 | 9 | ✔ | ✔ | Y | a-Arl8 | Y | 291 | 78 |
| Mitochondria | Tim17b | 3 | ✔ | ✔ | N | a-ATP5A | N | N | 266 |
| Mitochondria | Tom20 | 17 | ✔ | ✔ | Y | a-ATP5A | Y | 302 | 117 |
| Nuclear membrane, inner | dLBR | 0 | ✔ | N | N | N | N | N | 42 |
| Nuclear membrane, inner | Lamin | 53 | ✔ | ✔ | Y | a-Lamin | Y | 292 | 249 |
| Nucleolus | Fibrillarin | 14 | ✔ | ✔ | Y | a-Fib | Y | 278, 279 | 53 |
| Peroxisomes | Pmp70 | 6 | ✔ | ✔ | N | N | N | N | 26 |
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* = distribution was as expected, but no antibody available to test by co-stain
Additional files
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Supplementary file 1
Sequences of drop-in constructs.
- https://doi.org/10.7554/eLife.51539.015
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Supplementary file 2
Protocol for designing drop-in int100-CRIMIC constructs.
- https://doi.org/10.7554/eLife.51539.016
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Supplementary file 3
List of oligos and ultramers used in this study.
- https://doi.org/10.7554/eLife.51539.017
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Transparent reporting form
- https://doi.org/10.7554/eLife.51539.018
